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A leading pharmacological strategy toward HIV cure requires shock" or activation of HIV gene expression in latently infected cells with latency reversal agents (LRAs) followed by their subsequent clearance. In a screen for novel LRAs we used fungal secondary metabolites as a source of bioactive molecules. Using orthogonal mass spectrometry (MS) coupled to latency reversal bioassays we identified gliotoxin (GTX) as a novel LRA. GTX significantly induced HIV-1 gene expression in latent ex vivo infected primary cells and in CD4+ T cells from all aviremic HIV-1+ participants. RNA sequencing identified 7SK RNA the scaffold of the positive transcription elongation factor b (P-TEFb) inhibitory 7SK small nuclear ribonucleoprotein (snRNP) complex to be significantly reduced upon GTX treatment of CD4+ T cells. GTX directly disrupted 7SK snRNP by targeting La-related protein 7 (LARP7) releasing active P-TEFb which phosphorylated RNA polymerase II (Pol II) C-terminal domain (CTD) inducing HIV transcription." View Publication

Drug-induced resistance,or tolerance,is an emerging yet poorly understood failure of anticancer therapy. The interplay between drug-tolerant cancer cells and innate immunity within the tumor,the consequence on tumor growth,and therapeutic strategies to address these challenges remain undescribed. Here we elucidate the role of taxane-induced resistance on natural killer (NK) cell tumor immunity in triple-negative breast cancer (TNBC) and the design of spatio-temporally controlled nanomedicines,which boost therapeutic efficacy and invigorate 'disabled' NK. Drug tolerance limited NK cell immune surveillance via drug-induced depletion of the NK-activating ligand receptor axis,NKG2D and MHC class I polypeptide-related sequence A,B (MICA/B). Systems biology supported by empirical evidence revealed the heat shock protein 90 (Hsp90) simultaneously controls immune surveillance and persistence of drug-treated tumor cells. Based on this evidence,we engineered a 'chimeric' nano-therapeutic tool comprising taxanes and a cholesterol-tethered Hsp90 inhibitor,radicicol,which targets the tumor,reduces tolerance,and optimally re-primes NK cells via prolonged induction of NK-activating ligand receptors via temporal control of drug release in vitro and in vivo. A human ex-vivo TNBC model confirmed the importance of NK cells in drug-induced death under pressure of clinically-approved agents. These findings highlight a convergence between drug-induced resistance,the tumor-immune contexture,and engineered approaches that considers the tumor and microenvironment to improve the success of combinatorial therapy. View Publication

The recent approvals of anticancer therapeutic agents targeting the histone deacetylases and DNA methyltransferases have highlighted the important role that epigenetics plays in human diseases,and suggested that the factors controlling gene expression are novel drug targets. Protein arginine deiminase 4 (PAD4) is one such target because its effects on gene expression parallel those observed for the histone deacetylases. We demonstrated that F- and Cl-amidine,two potent PAD4 inhibitors,display micromolar cytotoxic effects towards several cancerous cell lines (HL-60,MCF7 and HT-29); no effect was observed in noncancerous lines (NIH 3T3 and HL-60 granulocytes). These compounds also induced the differentiation of HL-60 and HT29 cells. Finally,these compounds synergistically potentiated the cell killing effects of doxorubicin. Taken together,these findings suggest PAD4 inhibition as a novel epigenetic approach for the treatment of cancer,and suggest that F- and Cl-amidine are candidate therapeutic agents for this disease. View Publication

Thyromimetics that specifically target TR$\beta$ have been shown to reduce plasma cholesterol levels and avoid atherosclerosis through the promotion of reverse cholesterol transport in an animal model. We designed novel thyromimetics with high receptor (TR$\beta$) and organ (liver) selectivity based on the structure of eprotirome (3) and molecular modeling. We found that indane derivatives are potent and dual-selective thyromimetics expected to avoid hypothyroidism in some tissues as well as heart toxicity. KTA-439 (29),a representative indane derivative,showed the same high human TR$\beta$ selectivity in a binding assay as 3 and higher liver selectivity than 3 in a cholesterol-fed rat model. View Publication

PURPOSE PARP-3 is member of the PARP family of poly (ADP-ribose) polymerases involved in ADPribosylation. PARPs are involved in the basic mechanisms of DNA repair. PARP3,a critical player for efficient mitotic progression,is required for the stabilization of the mitotic spindle by regulation of the mitotic components,NuMA and Tankyrase 1. METHODS The sensitization effect of vinorelbine on PARP3 inhibition-induced cytotoxicity was assessed by the SRB assay. The contribution of programed cell death and cell cycle arrest to the sensitization effect were determined by assessing changes in Annexin V,a marker of apoptosis. Alterations in cell cycle progression were assessed by cell cycle analysis. We used immunofluorescence to assess the effect of vinorelbine and/or PARP3 inhibitors on tubulin and microtubule depolarization. The PARP3 chemiluminescent assay kit was used for PARP3 activity. RESULTS PARP3 inhibitors sensitize breast cancer cells to vinorelbine,a vinca alkaloid used in the treatment of metastatic breast cancer. Olaparib which was originally described as a PARP1 and 2 inhibitor has recently been shown to be a potent PARP3 inhibitor while ME0328 is a more selective PARP3 inhibitor. The combination of vinorelbine with nontoxic concentrations of ME0328 or olaparib reduces vinorelbine resistance by 10 and 17 fold,respectively,potentiating vinorelbine-induced arrest at the G2/M boundary. In addition,PARP3 inhibition potentiates vinorelbine interaction with tubulin. Furthermore,olaparib or ME0328 potentiates vinorelbine-induced PARP3 inhibition,mitotic arrest,and apoptosis. CONCLUSION Our results indicated this approach with PARP3 inhibitors and vinorelbine is unique and promising for breast cancer patients with metastases. This combination could significantly increase the survival of breast cancer patients with metastases. View Publication

The airway epithelium contains ionocytes,a rare cell type with high expression of Forkhead Box I1 (FOXI1) transcription factor and Cystic Fibrosis Transmembrane conductance Regulator (CFTR),a chloride channel that is defective in cystic fibrosis (CF). Our aim was to verify if ionocyte development is altered in CF and to investigate the relationship between ionocytes and CFTR-dependent chloride secretion. We collected nasal cells by brushing to determine ionocyte abundance. Nasal and bronchial cells were also expanded in vitro and reprogrammed to differentiated epithelia for morphological and functional studies. We found a relatively high ({\~{}}3{\%}) ionocyte abundance in ex vivo nasal samples,with no difference between CF and control individuals. In bronchi,ionocytes instead appeared very rarely as previously reported,thus suggesting a possible proximal-distal gradient in human airways. The difference between nasal and bronchial epithelial cells was maintained in culture,which suggests an epigenetic control of ionocyte development. In the differentiation phase of the culture procedure,we used two media that resulted in a different pattern of CFTR expression: confined to ionocytes or more broadly expressed. CFTR function was similar in both conditions,thus indicating that chloride secretion equally occurs irrespective of CFTR expression pattern. View Publication

BACKGROUND Nicotinamide phosphoribosyltransferase (NAMPT) is the key enzyme of the NAD salvage pathway starting from nicotinamide. Cancer cells have an increased demand for NAD due to their high proliferation and DNA repair rate. Consequently,NAMPT is considered as a putative target for anti-cancer therapies. There is evidence that AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) become dysregulated during the development of hepatocellular carcinoma (HCC). Here,we investigated the effects of NAMPT inhibition by its specific inhibitor FK866 on the viability of hepatocarcinoma cells and analyzed the effects of FK866 on the nutrient sensor AMPK and mTOR complex1 (mTORC1) signaling. RESULTS FK866 markedly decreased NAMPT activity and NAD content in hepatocarcinoma cells (Huh7 cells,Hep3B cells) and led to delayed ATP reduction which was associated with increased cell death. These effects could be abrogated by administration of nicotinamide mononucleotide (NMN),the enzyme product of NAMPT. Our results demonstrated a dysregulation of the AMPK/mTOR pathway in hepatocarcinoma cells compared to non-cancerous hepatocytes with a higher expression of mTOR and a lower AMPK$\alpha$ activation in hepatocarcinoma cells. We found that NAMPT inhibition by FK866 significantly activated AMPK$\alpha$ and inhibited the activation of mTOR and its downstream targets p70S6 kinase and 4E-BP1 in hepatocarcinoma cells. Non-cancerous hepatocytes were less sensitive to FK866 and did not show changes in AMPK/mTOR signaling after FK866 treatment. CONCLUSION Taken together,these findings reveal an important role of the NAMPT-mediated NAD salvage pathway in the energy homeostasis of hepatocarcinoma cells and suggest NAMPT inhibition as a potential treatment option for HCC. View Publication

Human regulatory T (Treg) cells are essential for immune homeostasis. The transcription factor FOXP3 maintains Treg cell identity,yet the complete set of key transcription factors that control Treg cell gene expression remains unknown. Here,we used pooled and arrayed Cas9 ribonucleoprotein screens to identify transcription factors that regulate critical proteins in primary human Treg cells under basal and proinflammatory conditions. We then generated 54,424 single-cell transcriptomes from Treg cells subjected to genetic perturbations and cytokine stimulation,which revealed distinct gene networks individually regulated by FOXP3 and PRDM1,in addition to a network coregulated by FOXO1 and IRF4. We also discovered that HIVEP2,to our knowledge not previously implicated in Treg cell function,coregulates another gene network with SATB1 and is important for Treg cell-mediated immunosuppression. By integrating CRISPR screens and single-cell RNA-sequencing profiling,we have uncovered transcriptional regulators and downstream gene networks in human Treg cells that could be targeted for immunotherapies. View Publication

Idiopathic pulmonary fibrosis (IPF) is a fatal disease of unknown cause that is characterized by progressive fibrotic lung remodeling. An abnormal emergence of airway epithelial-like cells within the alveolar compartments of the lung,herein termed bronchiolization,is often observed in IPF. However,the origin of this dysfunctional distal lung epithelium remains unknown due to a lack of suitable human model systems. In this study,we established a human induced pluripotent stem cell (iPSC)-derived air-liquid interface (ALI) model of alveolar epithelial type II (ATII)-like cell differentiation that allows us to investigate alveolar epithelial progenitor cell differentiation in vitro. We treated this system with an IPF-relevant cocktail (IPF-RC) to mimic the pro-fibrotic cytokine milieu present in IPF lungs. Stimulation with IPF-RC during differentiation increases secretion of IPF biomarkers and RNA sequencing (RNA-seq) of these cultures reveals significant overlap with human IPF patient data. IPF-RC treatment further impairs ATII differentiation by driving a shift toward an airway epithelial-like expression signature,providing evidence that a pro-fibrotic cytokine environment can influence the proximo-distal differentiation pattern of human lung epithelial cells. In conclusion,we show for the first time,the establishment of a human model system that recapitulates aspects of IPF-associated bronchiolization of the lung epithelium in vitro. View Publication

Pygopus 2 (Pygo2) is a coactivator of Wnt/$\beta$-catenin signaling that can bind bi- or trimethylated lysine 4 of histone-3 (H3K4me2/3) and participate in chromatin reading and writing. It remains unknown whether the Pygo2-H3K4me2/3 association has a functional relevance in breast cancer progression in vivo. To investigate the functional relevance of histone-binding activity of Pygo2 in malignant progression of breast cancer,we generated a knock-in mouse model where binding of Pygo2 to H3K4me2/3 was rendered ineffective. Loss of Pygo2-histone interaction resulted in smaller,differentiated,and less metastatic tumors,due,in part,to decreased canonical Wnt/$\beta$-catenin signaling. RNA- and ATAC-sequencing analyses of tumor-derived cell lines revealed downregulation of TGF$\beta$ signaling and upregulation of differentiation pathways such as PDGFR signaling. Increased differentiation correlated with a luminal cell fate that could be reversed by inhibition of PDGFR activity. Mechanistically,the Pygo2-histone interaction potentiated Wnt/$\beta$-catenin signaling,in part,by repressing the expression of Wnt signaling antagonists. Furthermore,Pygo2 and $\beta$-catenin regulated the expression of miR-29 family members,which,in turn,repressed PDGFR expression to promote dedifferentiation of wild-type Pygo2 mammary epithelial tumor cells. Collectively,these results demonstrate that the histone binding function of Pygo2 is important for driving dedifferentiation and malignancy of breast tumors,and loss of this binding activates various differentiation pathways that attenuate primary tumor growth and metastasis formation. Interfering with the Pygo2-H3K4me2/3 interaction may therefore serve as an attractive therapeutic target for metastatic breast cancer. SIGNIFICANCE: Pygo2 represents a potential therapeutic target in metastatic breast cancer,as its histone-binding capability promotes $\beta$-catenin-mediated Wnt signaling and transcriptional control in breast cancer cell dedifferentiation,EMT,and metastasis. View Publication

Cdc7-Dbf4 kinase or DDK (Dbf4-dependent kinase) is required to initiate DNA replication by phosphorylating and activating the replicative Mcm2-7 DNA helicase. DDK is overexpressed in many tumor cells and is an emerging chemotherapeutic target since DDK inhibition causes apoptosis of diverse cancer cell types but not of normal cells. PHA-767491 and XL413 are among a number of potent DDK inhibitors with low nanomolar IC50 values against the purified kinase. Although XL413 is highly selective for DDK,its activity has not been extensively characterized on cell lines. We measured anti-proliferative and apoptotic effects of XL413 on a panel of tumor cell lines compared to PHA-767491,whose activity is well characterized. Both compounds were effective biochemical DDK inhibitors but surprisingly,their activities in cell lines were highly divergent. Unlike PHA-767491,XL413 had significant anti-proliferative activity against only one of the ten cell lines tested. Since XL413 did not effectively inhibit DDK in multiple cell lines,this compound likely has limited bioavailability. To identify potential leads for additional DDK inhibitors,we also tested the cross-reactivity of ∼400 known kinase inhibitors against DDK using a DDK thermal stability shift assay (TSA). We identified 11 compounds that significantly stabilized DDK. Several inhibited DDK with comparable potency to PHA-767491,including Chk1 and PKR kinase inhibitors,but had divergent chemical scaffolds from known DDK inhibitors. Taken together,these data show that several well-known kinase inhibitors cross-react with DDK and also highlight the opportunity to design additional specific,biologically active DDK inhibitors for use as chemotherapeutic agents. View Publication

Thyroid hormone (TH) actions are mediated by nuclear receptors (TRs alpha and beta) that bind triiodothyronine (T(3),3,5,3'-triiodo-l-thyronine) with high affinity,and its precursor thyroxine (T(4),3,5,3',5'-tetraiodo-l-thyronine) with lower affinity. T(4) contains a bulky 5' iodine group absent from T(3). Because T(3) is buried in the core of the ligand binding domain (LBD),we have predicted that TH analogues with 5' substituents should fit poorly into the ligand binding pocket and perhaps behave as antagonists. We therefore examined how T(4) affects TR activity and conformation. We obtained several lines of evidence (ligand dissociation kinetics,migration on hydrophobic interaction columns,and non-denaturing gels) that TR-T(4) complexes adopt a conformation that differs from TR-T(3) complexes in solution. Nonetheless,T(4) behaves as an agonist in vitro (in effects on coregulator and DNA binding) and in cells,when conversion to T(3) does not contribute to agonist activity. We determined x-ray crystal structures of the TRbeta LBD in complex with T(3) and T(4) at 2.5-A and 3.1-A resolution. Comparison of the structures reveals that TRbeta accommodates T(4) through subtle alterations in the loop connecting helices 11 and 12 and amino acid side chains in the pocket,which,together,enlarge a niche that permits helix 12 to pack over the 5' iodine and complete the coactivator binding surface. While T(3) is the major active TH,our results suggest that T(4) could activate nuclear TRs at appropriate concentrations. The ability of TR to adapt to the 5' extension should be considered in TR ligand design. View Publication